Zinc Dynamics during Drosophila Oocyte Maturation and Egg Activation.

Temporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. Here, we demonstrate that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Our synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc.

Chemical-Biological Properties of Zinc Sensors TSQ and Zinquin: Formation of Sensor-Zn-Protein Adducts versus Zn(Sensor)2 Complexes.

Fluorescent zinc sensors are the most commonly used tool to study the intracellular mobile zinc status within cellular systems. Previously, we have shown that the quinoline-based sensors Zinquin and 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ) predominantly form ternary adducts with members of the Zn-proteome. Here, the chemistries of these sensors are further characterized, including how Zn(sensor)2 complexes may react in an intracellular environment. We demonstrate that these sensors are typically used in higher concentrations than needed to obtain maximum signal. Exposing cells to either Zn(Zinquin)2 or Zn(TSQ)2 resulted in efficient cellular uptake and the formation of sensor-Zn-protein adducts as evidenced by both a fluorescence spectral shift toward that of ternary adducts and the localization of the fluorescence signal within the proteome after gel filtration of cellular lysates. Likewise, reacting Zn(sensor)2 with the Zn-proteome from LLC-PK1 cells resulted in the formation of sensor-Zn-protein ternary adducts that could be inhibited by first saturating the Zn- proteome with excess sensor. Further, a native SDS-PAGE analysis of the Zn-proteome reacted with either the sensor or the Zn(sensor)2 complex revealed that both reactions result in the formation of a similar set of sensor-Zn-protein fluorescent products. The results of this experiment also demonstrated that TSQ and Zinquin react with different members of the Zn-proteome. Reactions with the model apo-Zn-protein bovine serum albumin showed that both Zn(TSQ)2 and Zn(Zinquin)2 reacted to form ternary adducts with its apo-Zn-binding site. Moreover, incubating Zn(sensor)2 complexes with non-zinc binding proteins failed to elicit a spectral shift in the fluorescence spectrum, supporting the premise that blue-shifted emission spectra are due to sensor-Zn-protein ternary adducts. It was concluded that Zn(sensors)2 species do not play a significant role in the overall reaction between these sensors and intact cells. In turn, this study further supports the formation of sensor-Zn-protein adducts as the principal observed fluorescent product during experiments employing these two sensors.

Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions.

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis. Although covalent structural features of resolved proteins can be determined with SDS-PAGE, functional properties are destroyed, including the presence of non-covalently bound metal ions. To address this shortcoming, blue-native (BN)-PAGE has been introduced. This method retains functional properties but at the cost of protein resolving power. To address the need for a high resolution PAGE method that results in the separation of native proteins, experiments tested the impact of changing the conditions of SDS-PAGE on the quality of protein separation and retention of functional properties. Removal of SDS and EDTA from the sample buffer together with omission of a heating step had no effect on the results of PAGE. Reduction of SDS in the running buffer from 0.1% to 0.0375% together with deletion of EDTA also made little impact on the quality of the electrophoretograms of fractions of pig kidney (LLC-PK1) cell proteome in comparison with that achieved with the SDS-PAGE method. The modified conditions were called native (N)SDS-PAGE. Retention of Zn(2+) bound in proteomic samples increased from 26 to 98% upon shifting from standard to modified conditions. Moreover, seven of nine model enzymes, including four Zn(2+) proteins that were subjected to NSDS-PAGE retained activity. All nine were active in BN-PAGE, whereas all underwent denaturation during SDS-PAGE. Metal retention after electrophoresis was additionally confirmed using laser ablation-inductively coupled plasma-mass spectrometry and in-gel Zn-protein staining using the fluorophore TSQ.

Glutathione-mediated neuroprotection against methylmercury neurotoxicity in cortical culture is dependent on MRP1.

Methylmercury (MeHg) exposure at high concentrations poses significant neurotoxic threat to humans worldwide. The present study investigated the mechanisms of glutathione-mediated attenuation of MeHg neurotoxicity in primary cortical culture. MeHg (5 µM) caused depletion of mono- and disulfide glutathione in neuronal, glial and mixed cultures. Supplementation with exogenous glutathione, specifically glutathione monoethyl ester (GSHME) protected against the MeHg induced neuronal death. MeHg caused increased reactive oxygen species (ROS) formation measured by dichlorodihydrofluorescein (DCF) fluorescence with an early increase at 30 min and a late increase at 6h. This oxidative stress was prevented by the presence of either GSHME or the free radical scavenger, trolox. While trolox was capable of quenching the ROS, it showed no neuroprotection. Exposure to MeHg at subtoxic concentrations (3 µM) caused an increase in system x(c)(-) mediated (14)C-cystine uptake that was blocked by the protein synthesis inhibitor, cycloheximide (CHX). Interestingly, blockade of the early ROS burst prevented the functional upregulation of system x(c)(-). Inhibition of multidrug resistance protein-1 (MRP1) potentiated MeHg neurotoxicity and increased cellular MeHg. Taken together, these data suggest glutathione offers neuroprotection against MeHg toxicity in a manner dependent on MRP1-mediated efflux.

Reactions of the fluorescent sensor, Zinquin, with the zinc-proteome: adduct formation and ligand substitution.

Zinquin (ZQ) is a commonly used sensor for cellular Zn(2+) status. It has been assumed that it measures accessible Zn(2+) concentrations in the nanomolar range. Instead, this report shows a consistent pattern across seven mammalian cell and tissue types that ZQ reacts with micromolar concentrations of Zn(2+) bound as Zn-proteins. The predominant class of products were ZQ-Zn-protein adducts that were characterized in vivo and in vitro by a fluorescence emission spectrum centered at about 470 nm, by their migration over Sephadex G-75 as protein not low molecular weight species, by the exclusion of reaction with lipid vesicles, and by their large aggregate concentration. In addition, variable, minor formation of Zn(ZQ)(2) with a fluorescence band at about 490 nm was observed in vivo in each case. Because incubation of isolated Zn-proteome with ZQ also generated similar amounts of Zn(ZQ)(2), it was concluded that this species had formed through direct ligand substitution in which ZQ had successfully competed for protein-bound Zn(2+). Parallel studies with the model Zn-proteins, alcohol dehydrogenase (ADH), and alkaline phosphatase (AP) revealed a similar picture of reactivity: ZQ(ACID) (Zinquin acid, (2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate)) able to bind to one Zn(2+) and extract the other in Zn(2)-ADH, whereas it removed one Zn(2+) from Zn(2)-AP and did not bind to the other. Zinquin ethyl ester (ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate); ZQ(EE)) bound to both proteins without sequestering Zn(2+) from either one. In contrast to a closely related sensor, 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ), neither ZQ(ACID) nor ZQ(EE) associated with Zn-carbonic anhydrase. A survey of reactivity of these sensors with partially fractionated Zn-proteome confirmed that ZQ and TSQ bind to distinct, overlapping subsets of the Zn-proteome.